The anatomical, molecular and functional properties that characterize different stages of differentiation of the mammalian olfactory receptor neurons (ORNs) are not well understood. In addition, little is known about the control systems regulating movement from one differentiation stage to another. To characterize ORN differentiation stages and identify regulatory signals, we have developed dissociated cell culture systems of newborn and adult rat nasal cells. In these cultures, ORNs are generated and undergo both initial differentiation and maturation (electrical responses to odorants and expression of the olfactory marker protein). This is the only dissociated culture system described to date that shows maturation of ORNs. Our preliminary data have shown or suggested that ORNs have several stages of differentiation, that neurogenesis and differentiation are under separate regulatory control, and that specific bulb-epithelial, neuron/neuron contacts are not necessary for ORN maturation in vitro. We propose herein to use these dissociated cell cultures, in the first half of this grant, to further characterize the stages of olfactory neuronal differentiation. We will analyze anatomical and molecular changes in ORNs with immunocytochemistry, electron microscopy and in situ hybridization, in collaborations with several members of this Program Project Grant (PPG) group. We will characterize stages in the development of ORN odorant responses with the voltage sensitive dye technology and correlate these stages with stages in molecular differentiation. In the second half of this grant, we propose to investigate specific regulatory controls of differentiation stages, again in collaboration with members of the PPG group. In particular, we propose to investigate the hypothesis that neuronal contact between ORNs and olfactory bulb neurons in culture will provide an additional level of trophic support to ORNs, resulting in an increase in the life span of ORNs and greater numbers of mature, olfactory marker, protein-positive ORNs. Then, we will use the dissociated cell cultures to characterize the effects of specific growth factors on neuronal genesis and initial differentiation into neurons. The EGF family of growth factors has been implicated in control of olfactory neurogenesis and the TGFbeta family has been implicated in initial olfactory neuronal differentiation. These studies will provide information on basic mechanisms of neuronal differentiation that will be instructive of the function of the nervous system in general. Furthermore, knowledge gained about the regulation of olfactory neuronal generation and differentiation might be applicable to other neuronal populations where replacement and continual differentiation and maturation of neurons do not occur.